Characterization of atypical Aeromonas salmonicida isolates by ribotyping and plasmid profiling

A total of 38 strains of atypical Aeromonas salmonicida , three oxidase-negative but otherwise typical Aer. salmonicida , three typical Aer. salmonicida , and two reference strains, isolated from several countries and fish species were examined with respect to rRNA gene restriction patterns (ribotypes) and plasmid profiles. Most epidemiologically unrelated strains had different ribotypes, whereas isolates from the same outbreak were identical. All strains, except one, carried one or more large plasmids (> 55 kbp) and all strains, except two, additionally carried one or more smaller plasmids. Many strains isolated from the same outbreak showed different plasmid profiles although some plasmids were identical. The results suggest the existence of several atypical Aer. salmonicida. It also seems that ribotypes are stable properties for these bacteria while the plasmids are more labile.     

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.     

Insight into the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to antimicrobial drugs analysed by the 454-pyrosequencing technology

Wastewater treatment plants (WWTPs) are a reservoir for bacteria harbouring antibiotic resistance plasmids. To get a comprehensive overview on the plasmid metagenome of WWTP bacteria showing reduced susceptibility to certain antimicrobial drugs an ultrafast sequencing approach applying the 454-technology was carried out. One run on the GS 20 System yielded 346,427 reads with an average read length of 104 bases resulting in a total of 36,071,493 bases sequence data. The obtained plasmid metagenome was analysed and functionally annotated by means of the Sequence Analysis and Management System (SAMS) software package. Known plasmid genes could be identified within the WWTP plasmid metagenome data set by BLAST searches using the NCBI Plasmid Database. Most abundant hits represent genes involved in plasmid replication, stability, mobility and transposition. Mapping of plasmid metagenome reads to completely sequenced plasmids revealed that many sequences could be assigned to the cryptic pAsa plasmids previously identified in Aeromonas salmonicida subsp. salmonicida and to the accessory modules of the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas punctata. Matches of sequence reads to antibiotic resistance genes indicate that plasmids from WWTP bacteria encode resistances to all major classes of antimicrobial drugs. Plasmid metagenome sequence reads could be assembled into 605 contigs with a minimum length of 500 bases. Contigs predominantly encode plasmid survival functions and transposition enzymes.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

Bacterial Plasmids in Antarctic Natural Microbial Assemblages

Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica.     

Control of Plasmid R1 Replication: Functions Involved in Replication, Copy Number Control, Incompatibility, and Switch-off of Replication

A small derivative of plasmid R1 was used to integratively suppress a chromosomal dnaA(Ts) mutation. The strain obtained grew normally at 42°C. The integratively suppressed strain was used as recipient for various plasmid R1 derivatives. Plasmid R1 and miniplasmid derivatives of R1 could be established in the strain that carried an integrated R1 replicon, but they were rapidly lost during growth. However, plasmids also carrying ColE1 replication functions were almost completely stably inherited. The integratively suppressed strain therefore allows the establishment of bacteria diploid with respect to plasmid R1 and forms a useful and sensitive system for studies of interaction between plasmid R1 replication functions. Several of the chimeric plasmids caused inhibition of growth at high temperatures. All plasmids that inhibited growth carried one particular PstI fragment from plasmid R1 (the PstI F fragment), and in all cases the growth inhibition could be ascribed to repression of initiation of chromosome replication at 42°C, i.e., they carry a trans-acting switch-off function. Furthermore, the analogous PstI fragments from different copy mutants of plasmid R1 were analyzed similarly, and one mutant was found to lack the switch-off function. The different chimeric plasmids were also tested for their incompatibility properties. All plasmids that carried the switch-off function (and no other plasmids) also carried R1 incompatibility gene(s). Since the PstI F fragment, which is present on all these plasmids, is very small (0.35 × 10⁶), it is suggested that the switch-off regulation of replication (by an inhibitor), incompatibility, and copy number control are governed by the same gene.     

大肠埃希菌耐药性监测及耐药质粒的研究

为了解医院内感染大肠埃希菌的耐药情况,探讨其耐药发生、流行及传播机制,对从临床分离的32株大肠埃希菌进行了药敏试验、质粒图谱分析以及质粒接合、转化试验,结果表明大肠埃希菌对氨苄青霉素的耐药率＞88%,对庆大霉素的耐药率＞75%,其中28株大肠埃希菌检出质粒,均含有一条分子量为5.66 Mu的质粒带,是医院内感染大肠埃希菌的流行质粒.质粒的接合、转化试验证实了质粒具有横向传播的特点,是细菌产生耐药的主要原因.     

Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells.

We have constructed plasmids carrying direct internal repeats 260-2000 bp long. Monomers of such plasmids transformed Bacillus subtilis competent cells. The efficiency of transformation varied with the square of the length of repeats. The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid. Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats. When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur. The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.     

Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection.

In an outbreak of hospital infection caused by Klebsiella aerogenes type K-16 isolates over a 3-month period carried, apparently unaltered, a cryptic 90-Megadalton (Md) plasmid (unclassified) and a multiple-resistance 65-Md plasmid of IncM. The IncM plasmid, identified in environmentally related strains of Citrobacter koseri and Escherichia coli, showed minor variations from that in the klebsiella vector. The IncM plasmids, as well as all wild host strains cured of the IncM plasmids, carried a transposable DNA sequence, encoding trimethoprim and, in every case but one, streptomycin resistance. This transposon appeared identical with Tn7, previously identified in unrelated plasmids in bacteria from different environments.     

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids. This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid. Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies. These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA.     

Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.     

Self-Transmissible Mercury Resistance Plasmids with Gene-Mobilizing Capacity in Soil Bacterial Populations: Influence of Wheat Roots and Mercury Addition

A set of mercury resistance plasmids was obtained from wheat rhizosphere soil amended or not amended with mercuric chloride via exogenous plasmid isolation by using Pseudomonas fluorescens R2f, Pseudomonas putida UWC1, and Enterobacter cloacae BE1 as recipient strains. The isolation frequencies were highest from soil amended with high levels of mercury, and the isolation frequencies from unamended soil were low. With P. putida UWC1 as the recipient, the isolation frequency was significantly enhanced in wheat rhizosphere compared to bulk soil. Twenty transconjugants were analyzed per recipient strain. All of the transconjugants contained plasmids which were between 40 and 50 kb long. Eight selected plasmids were distributed among five groups, as shown by restriction digestion coupled with a similarity matrix analysis. However, all of the plasmids formed a tight group, as judged by hybridization with two whole-plasmid probes and comparisons with other plasmids in dot blot hybridization analyses. The results of replicon typing and broad-host-range incompatibility (Inc) group-specific PCR suggested that the plasmid isolates were not related to any previously described Inc group. Although resistance to copper, resistance to streptomycin, and/or resistance to chloramphenicol was found in several plasmids, catabolic sequences were generally not identified. One plasmid, pEC10, transferred into a variety of bacteria belonging to the β and γ subdivisions of the class Proteobacteria and mobilized as well as retromobilized the IncQ plasmid pSUP104. A PCR method for detection of pEC10-like replicons was used, in conjunction with other methods, to monitor pEC10-homologous sequences in mercury-polluted and unpolluted soils. The presence of mercury enhanced the prevalence of pEC10-like replicons in soil and rhizosphere bacterial populations.The potential use of genetically modified bacteria in agriculture has raised questions pertaining to the spread of introduced recombinant DNA through soil bacterial communities. Gene transfer in soil via conjugation has received much attention, and the focus of most studies has been the transfer and fate of introduced plasmids (6, 22, 27–29, 39). Under favorable conditions, in specific soil microhabitats, or under selection conditions, both self-transmissible and mobilizable plasmids present in introduced hosts can be transferred to introduced recipients, as well as to a variety of indigenous bacteria (15, 20, 27, 28, 33). In particular, rhizospheres of crop plants, such as wheat and sugar beet, provide conditions conducive to conjugal plasmid transfer between bacterial inhabitants (15, 36). When genetically modified bacteria are developed as inoculants for the rhizosphere, insertion of heterologous DNA into non-self-transmissible plasmids or the chromosome might restrict conjugal transfer of this DNA to members of the indigenous bacterial community. However, mobilizing or retromobilizing (33) plasmids present in indigenous soil bacteria could potentially still effect the transfer of the less mobile heterologous DNA via chromosome or plasmid mobilization, which may involve cointegration (9, 19, 31). Such plasmids might thus be responsible for the escape of heterologous DNA from genetically modified bacteria introduced into soil.There is a paucity of knowledge concerning the incidence of plasmids with mobilizing capacity in soils and rhizospheres, as well as concerning the effects of soil factors, such as stresses resulting from pollution or from natural causes (e.g., rhizosphere acidity), on plasmid prevalence and transfer (e.g., reference 38). Whereas it has been suggested that chemical stress often does not enhance plasmid incidence in selected soil bacterial populations (40), pollution in river water or mines (in particular mercury pollution) has been found to exert a selective (enhancing) effect (4, 13).Plasmids of environmental bacteria have classically been obtained by endogenous isolation procedures (20). Endogenous isolation implies that putative plasmid hosts with the phenotype of interest are isolated from soil, after which plasmids are extracted from pure cultures of these strains. On the other hand, pioneering studies performed with river stone epilithon (9) and later extended to soil and sediment (32) have shown that plasmids can be obtained directly from indigenous bacterial communities in new hosts by exogenous isolation. In this approach, plasmids are captured in selectable recipient strains by using mating between these strains and the total bacterial community obtained from an environmental sample. Following incubation, the mating mixture is plated with selection for the recipient and an additional marker gene presumedly located on a plasmid present in the indigenous bacteria (6). The advantage of the exogenous isolation procedure is that no culturing step is required in the mating, which thus allows isolation of plasmids from nonculturable hosts. Furthermore, plasmids are directly selected for their transfer capacity, in addition to the presence of a specific selectable marker.In this study, exogenous plasmid isolation was employed to obtain transferable plasmids from soil bacteria by using mercury resistance as the selectable marker. The objective of this work was to gain insight into the potential present in soil bacterial populations to (retro)mobilize genes out of introduced bacteria into members of the soil bacterial community. Since the incidence of plasmids in soil bacteria is likely influenced by soil ecological factors and selection pressure, the presence of wheat roots and selection by mercury (25) were studied as experimental variables.     

Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2

Genes encoding toxin-antitoxin proteins are frequently found on plasmids where they serve to stabilize the plasmid within a bacterial population. The toxin-antitoxin proteins do not increase the likelihood of a progeny cell receiving a plasmid but rather function as post-segregational killing mechanisms which decrease the proportion of cells that survive after losing the plasmid. These toxin-antitoxin couples therefore act as plasmid addiction systems. Several new proteic toxin-antitoxin systems have been identified and these systems appear to be ubiquitous on the chromosomes of bacteria and archaea. When placed on plasmids, these chromosomal systems also have the ability to stabilize plasmids and in at least one case, chromosomal- and plasmid-based toxin-antitoxin systems have been shown to interact. Recent findings regarding toxin-antitoxin systems and questions that have arisen as a result of these findings are reviewed.     

Plasmid-linked Resistance to Inorganic Salts in Staphylococcus aureus

The penicillinase plasmids, a series of extrachromosomal resistance factors in Staphylococcus aureus, were found to carry determinants of resistance to a series of inorganic ions as well as resistance to penicillin and, in some cases, erythromycin. Most of the ions involved were inhibitory but not lethal to the bacteria; the resistance markers conferred an increase in resistance by comparison with susceptible organisms of between 3- and 100-fold, depending on the ion involved. Separate genetic loci for resistance to arsenate, arsenite, lead, cadmium, mercuric, and bismuth ions were demonstrated. Resistance to antimony and resistance to zinc were also found but were not separated genetically from resistance to arsenite and cadmium, respectively. The ion resistance markers appeared to form a cluster on the plasmid, with no other known marker within it. Naturally occurring plasmids were observed that lacked one or more of these ion resistance markers, as well as penicillinase-negative strains that were resistant to one or more of the ions. The patterns of markers carried by these various strains may provide some understanding of the evolution of a plasmid linkage group.     

Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum

A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRleVF39a, carries genes for the production of a melanin-like pigment as has been previously reported. Plasmid pRleVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix- nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRleVF39c contained few, if any, bacteria. Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.     

Characterization and cloning of plasmids from the iron-oxidizing bacteriumThiobacillus ferrooxidans

More than 100 independent strains ofThiobacillus ferrooxidans were isolated from six different domestic mining sites. Although there was some variation according to sampling site, about 73% of all strains carried more than one plasmid ranging in size from about 2.0 to 30 kilobase-pairs(kb). Among these, four plasmids of 2.4, 4.7, 5.1, and 8.9 kb, designated pTNA33, pTSY91, pTSB121, and pTSB122, respectively, were cloned intoEscherichia coli plasmids. pTSB121 and pTSB122, originated from the sameT. ferrooxidans strain, showed weak homology by Southern blotting, whereas pTSB121 showed high homology with pTSY91 from a different strain. It seems that the occurrence of the plasmid homologous to pTSB121 or pTSB122 is more ubiquitous inThiobacillus. On the other hand, pTNA33 is a unique plasmid because it showed no significant homology with other plasmids.     

Screening of plasmids in non-pathogenic corynebacteria

Abstract A screening of plasmids in 25 nonpathogenic coryneform bacteria was carried out. 11 Strains showed at least one plasmid, ranging in size from 4.2 to 55 kb. These plasmids did not encode bacteriocin production or resistance to a number of antibiotics or to ions such as arsenite, mercury(II) and cobalt(II). A detailed study of plasmid pBL100 from Brevibacterium linens is presented. pBL100 has a size of 7.75 kb, and contains single sites for the endonucleases: Hin dIII; Pst I, Bgl II, Eco RI and Bam HI. B. linens is easily and efficiently transformed with vectors derived from pBL1 isolated from Brevibacterium lactofermentum .